Defective regulation of in vivo hormone and neurotransmitter levels is associated with a wide range of human disorders, including diabetes, depression, and numerous others. Quantification of hormone and neurotransmitter secretion from single cells has provided key insights into the dynamics and mechanisms of cell signaling processes regulated by these molecules. Carbon fiber microelectrodes have been widely used in these studies to enable rapid, highly sensitive, and label-free measurement of several electroactive neurotransmitters (dopamine, serotonin, norepinephrine, etc.), as well as a few hormones (e.g. insulin). These studies have yielded key information regarding the signaling mechanisms associated with a range of disorders including depression, addiction, and diabetes. Unfortunately, the number of cellular secretory products that can be assayed via voltammetric and amperometric methods is highly limited, with less than 10 targets commonly analyzed. Developing a capability to rapidly detect a wider range of hormones and neurotransmitters in a label-free manner, with high sensitivity, selectivity and temporal resolution but minimal sample preparation, would enable investigation of a larger catalog of hormones and neuromodulators, and would establish a new paradigm in studies of cell signaling. We propose to develop a novel class of biochemical sniffer sensors in which a recombinant protein chimera, comprised of an ion channel conjugated to a G-protein coupled receptor (referred to as an ICCR), is reconstituted into an ultrastable planar membrane, and composed of a polymerized phospholipid bilayer that is suspended across a glass micropipet. Upon ligand binding and conformational activation of the GPCR subunit of the ICCR, ion flux through the IC subunit will be modulated and subsequently measured via electrophysiological detection. The sensitivity will be comparable to the Kd for the ligand-receptor pair, typically in the nM- M regime. Thus this technology will extend label-free, electrophysiological detection to cell signaling molecules that lack optical or electrochemical activity. During the project period: a) ICCRs will be prepared, purified, and reconstituted into artificial lipid membranes that are optimized for stability and retention of protein activity. b) Key structure-activity relationships of ICCRs in artificial membranes will be examined, such as the orientation and stoichiometry of the IC and GPCR subunits in functional ICCRs, and this information will be used to design more responsive ICCR transducers. c) Sensors based on ICCRs containing the D2 dopamine and glucagon receptors will be prepared and evaluated for selectively measuring dopamine and glucagon secretion, respectively, and then implemented to monitor stimulated release of these targets from single cells. Successful completion of this work will provide a powerful new tool for quantitative detection of biologically important yet analytically challenging cellular releasates.